Apparatus for sedimentation based blood analysis

ABSTRACT

An apparatus and process for accurately determining settling data for the settling of erythrocyte cells from a plasma fluid in a test specimen of blood. The apparatus includes a settling tube, a sensing assembly movably mounted proximate the settling tube. Preferably an infrared emitter and detector are provided in the sensing assembly, and a control assembly is provided which senses data at a high rate and is responsive to the sensed data to sample or store the time at which sensed reflectivity exceeds a threshold level. When the threshold is reached, data is sampled and the tracking head is moved by a very small step. This process is repeated to enable tracking of the descent of the separation boundary between the erythrocyte cells and plasma fluid. The apparatus senses changes in reflectivity of the erythrocyte portion of the specimen below and up to the separation boundary. The apparatus and process also includes a scanning process to calibrate the reflectivity for each test specimen, the reflectivity also is measured immediately after moving the sensing assembly and recorded as a function of the position of the sensing assembly. An apparatus and process for scanning the plasma fluid and white cells above the separation boundary for reflectivity of infrared or visible light radiation as a function of height above the separation boundary also is disclosed.

This is a division, of application Ser. No. 08/259,018 filed on Jun. 13,1994, now U.S. Pat. No. 5,487,870, which was a division of applicationSer. No. 07/512,845 filed on Apr. 23, 1990, which is now issued U.S.Pat. No. 5,328,822.

TECHNICAL FIELD

The present invention relates, in general, to apparatus and methods forthe analysis of blood specimens, and more particularly, relates toapparatus and methods for sedimentation-based analysis of bloodspecimens and the correlation of such analysis with inflammatoryconditions.

BACKGROUND ART

Since at least the 1920s, the analysis of blood based upon thesedimentation or separation of erythrocyte cells from blood plasma hasbeen extensively undertaken. Two classic sedimentation techniques aremost commonly employed, namely, the Wintrobe method and the Westergrenmethod. These techniques each utilize observation and recording of thesettling of erythrocyte or red cells in a blood specimen from therelatively clear white cell-containing plasma fluid. As the red cellssettle, a separation boundary between the erythrocyte cells and clearplasma occurs and drops or moves down the tube with continued red cellsettling.

During settling, the erythrocyte cells periodically stack in structuresknown as "rouleaux" and start to gravitate through the fluid plasmatoward the bottom of the settling tube. The relative movement betweenthe rouleaux and plasma causes the rouleaux to breakdown and setting tobe slowed. This process is repeated with time and affects the rate atwhich the separation boundary settles. The amount or drop of theseparation boundary in a predetermined time interval, for example onehour, has been referred to as the "settling rate" of the blood sample.This settling rate has been found to have a significant, although notvery specific, correlation with the presence of inflammatory diseasesand/or conditions.

While classic sedimentation rate measurements provide some usefulinformation as to the presence or absence of inflammatory conditions ina patient, they inherently have been incapable of specificallyidentifying conditions or diseases, and they have not been very usefulin tracking the progress of, or degree of involvement in, the disease.

Attempts have been made to gain more useful data from blood specimensettling or sedimentation. Additionally, various apparatus have beenemployed to attempt to automate the sensing and recording of erythrocytecell sedimentation. The attempts to automate the classic sedimentationrate measurement techniques have generally proceeded along the same lineas the original manual approach, namely, to employ an apparatus whichtransmits radiation, usually radiation in the visible light range,through the test specimen. Various recording and tracking heads havebeen employed in which the movement of the head occurs when there is achange from an inability to transmit light through the sample to anability to transmit light. This change occurs when the separationboundary drops down below the position of the light beam emitting andsensing apparatus. Typical of such blood sedimentation apparatus andmethods are the devices shown in U. S. Pat. Nos. 2,725,782, 2,982,170,3,261,256, 3,288,019, 3,422,443, 3,474,458, 3,604,924, 3,631,513,3,715,761, 3,844,662, 3,952,579 and 4,118,974.

It has been recognized that a single measurement taken one hour or twohours after the specimen is placed in the settling tube only providesone or two points on the "settling curve". Thus, the goal of automatingthe recording of sedimentation of erythrocyte cells was in part todevelop an entire settling curve in the hope that the curve wouldprovide more useful data that could be correlated to inflammatoryconditions.

Study of the sedimentation curves produced by various continuousrecording apparatus, however, has revealed that accuracy in thecorrelation of inflammatory conditions with settling curve shapes hasnot been possible. Diagnosis of inflammatory conditions using thecomplete settling curve for erythrocyte cells over a one, or even a twohour, settling period has lacked the reliability necessary for a sounddiagnosis.

The suggestion is also found in the prior art that the instantaneousrate of settling, or the derivative of the settling curve (change inheight divided by the change in time), may yield data which are morehelpful and more easily correlated to permit diagnosis of inflammatoryconditions than the settling curve itself. Thus, in U.S. Pat. No.4,041,502 to Williams et al. and a companion article entitled "AnAutomatic Sedimentimeter" in Biorheoloqy (Vol. 14, pp. 145-149, 1977)Misiaszek, Williams, Stasiw and Cerny, an apparatus and method fordiscrete recording of both the settling curve and the first derivativeor instantaneous settling rate of the settling curve are disclosed.

In the sedimentimeter of the Williams et al. patent, a light-emittingdiode is mounted next to a vertical settling tube on a movable trackinghead. The LED transmits a light beam through the tube to a photodiodedetector positioned on the tracking head on the other side of thespecimen. When the photodiode detector cannot detect the beam from theLED, the tracking head is below the separation boundary between theerythrocyte cells and fluid plasma. As the separation boundary drops toa position below the beam from the LED, the photodiode detector detectsthe beam and thereby senses the passage of the separation boundarybeyond the current beam position.

The Williams et al. tracking head is moved down the tube by a steppingmotor. The LED emits static or constant radiation which continuouslyirradiates the photodiode through the tube, and if the photodiodedetector senses light from the LED above a predetermined thresholdlevel, the stepping motor is actuated to drop the tracking head. Thesystem includes a clock and logic circuit which operates the steppingmotor for a period of time sufficient to cause the amount of lightsensed by the photodiode detector to drop below a certain level, atwhich point the stepping motor ceases operation. A recording circuitsamples data as to the time which the motor has operated every 15seconds and records the length of time of operation of the steppingmotor for each 15 second interval to thereby provide a record of themotion of the tracking head and the separation boundary.

In order to obtain further data for correlation with diseases, in theWilliams et al. patent the approximate slope of the sedimentation curveor the instantaneous rate of sedimentation also is calculated. This isaccomplished by determining the drop in distance of the tracking headfor each 15 second interval of time over the entire settling period,usually one hour.

The instantaneous settling rate or first derivative of the settlingcurve which is produced by the Williams et al. apparatus was thought toproduce data that would be more likely to be able to be correlated withinflammatory conditions. For example, the first derivative or slopecurve might yield information as to the formation and breakdown ofrouleaux, which may be an indication of the presence or absence of aninflammatory condition. Thus far, however, the hoped-for correlation ofthe settling rate data with inflammatory conditions has not beenrealized, and erythrocyte cell sedimentation studies still have not beenproven to be capable of reliable disease diagnosis.

Other articles in the technical literature since the Williams et al.patent which discuss erythrocyte cell sedimentation and the continuinglimited usefulness of sedimentation rates as a diagnostic tool include:

"The Age-Related Hemorheological and Osmotic Properties of Human Blood,"by Cerny et al., Biorheology, Vol. 74, No. 182, pp. 85-89 (1978);

"The Erythrocyte Sedimentation Rate of Blood Reconsidered," by Merrillet al., Biorheology, Vol. 74, No. 182, pp. 90-95 (1978);

"Optical Method for Haematocrit Determination," by Singh et al., Medical& Biological Engineering & computing, vol. 20, pp. 527-528 (July, 1982);

"The Erythrocyte Sedimentation Rate Time Curve: Critique of anEstablished Solution," by Dorrington et al., Biomechanics, Vol. 16, No.1, pp. 99-100 (1983); and

"Erythrocyte Sedimentation Rate--From Folklore to Facts," by Bedell etal., The American Journal of Medicine, Vol. 78, pp. 1001-1009 (June,1985).

The white cell-containing plasma above the settled erythrocyte cells haslargely been ignored as a source of usable sedimentation-based data. Inpatients having a significant one-hour erythrocyte settling rate, forexample, over about 7 millimeters, however, bands or layers of whitecells can be seen to occur above the settled red cells. This white cellbanding suggests that sedimentation is occurring in connection with thedifferent types of white cells. While various clinical tests have beendevised for white cells, they have not previously included attempts toobtain data useful in the diagnosis of inflammatory conditions fromwhite cells based upon their settling and banding characteristics.

Accordingly, it is an object of the present invention to provide aprocess and apparatus which is capable of a sufficiently accuratedetermination of the instantaneous settling rate of erythrocyte cells toenable the diagnosis of specific inflammatory conditions.

Another object of the present invention is to provide an erythrocytesedimentation tracking process and apparatus in which data is sampledwhen changes in the cell settling rate actually occur, rather than attimed intervals, to enable sensing of the occurrence of changes insedimentation.

Another object of the present invention is to provide an erythrocytesedimentation tracking process in which changes in red cellsedimentation which are affected by rouleaux formation and rouleauxbreakdown, and protein-protein interaction can be recorded and used tocorrelate settling with inflammatory conditions.

A further object of the present invention is to provide an erythrocytesedimentation-based process for diagnosing inflammatory conditions inwhich changes in settling characteristics of erythrocyte cells as theysettle past a sensing zone are determined.

Still another object of the present invention is to provide anerythrocyte sedimentation instrument and process which is more sensitiveto changes in settling rates and provides more data for the statisticalanalysis necessary for correlation to inflammatory conditions.

A further object of the present invention is to porvide an instrumentand process which senses and samples a characteristic (preferablyreflectivity) of settling of fluid plasma containing white cells andenables correlation of a sampled fluid plasma characteristic toinflammatory conditions.

Another object of the present invention is to provide an apparatus andprocess for the tracking of instantaneous settling rates of erythrocytecells which is self-calibratable to each test specimen of blood forimproved accuracy and higher correlation of instantaneous sedimentationrates with inflammatory conditions.

Still a further object of the present invention is to provide a processfor the use of repetitive patterns occurring in one or all of:erythrocyte instantaneous settling rates, white cell reflectivity scans,erythrocyte reflectivity scans, and erythrocyte reflectivity immediatelyafter a settling increment occurs, in the identification of inflammatoryconditions.

Another object of the present invention is to provide a blood cellsedimentation apparatus which is relatively simple and inexpensive toconstruct, operate and maintain.

A further object of the present invention is to provide a cellsedimentation apparatus which is capable of obtaining useful data fromboth red cell and white cell sedimentation.

Still another object of the present invention is to provide a method forobtaining useful red and white cell sedimentation data which can beautomated and produces data suitable for computer analysis and computercorrelation to inflammatory conditions.

The apparatus and process of the present invention have other objectsand features of advantage which will become apparent from or are setforth in more detail in, the accompanying drawing and the followingdescription of the Best Mode of Carrying Out the Invention.

DISCLOSURE OF INVENTION

The present apparatus for determining settling-based phenomena of bothred and white cells in a test specimen of blood includes a settlingtube, a sensing assembly mounted proximate the settling tube for sensinga characteristic of the blood cells and/or plasma during settling, acontrol assembly responsive to the sensing assembly to cause the sensingassembly to be displaced during sensing and sampling, and a datasampling apparatus responsive to at least one of the sensing assemblyand the control assembly to sample data indicating settling of the bloodcells.

The improvement in one aspect of the present invention is to provide anapparatus and method which yields data as to the settling of erythrocytecells from white cell-containing fluid plasma, which data can be moreaccurately correlated with inflammatory conditions. Thus, in theimproved apparatus and method the sensing assembly is operated to sensea desired characteristic, preferably reflectivity, at a sensing ratewhich is high relative to the rate of occurrence of a significantsettling of erythrocyte cells, for example about 50 times faster thanthe red cell settling rate. Moreover, in the improved apparatus andprocess sensed data is sampled, i.e., stored and used, only upon theoccurrence of a significant settling of the erythrocyte cells, forexample, upon settling of cells by an amount which will cause the sensedcharacteristic to cross a predetermined threshold. In this aspect of theinvention settling rate curves are generated using data sampling attimes which are determined by settling activity, not arbitrary timeintervals.

In another aspect, the improvement in the instantaneous erythrocytesettling rate apparatus and method of the present invention iscomprised, briefly, of the control assembly being responsive to thesensing assembly to maintain the sensing assembly located in a movabletracking zone during settling, which tracking zone is located from aposition proximate and below the separation boundary to a position up tothe separation boundary. Thus, sensing and sampling occurs by sensing achange in a characteristic of the test specimen in a tracking zonecontaining the erythrocyte cells, rather than in the fluid plasmaportion of the specimen. Moreover, sensing preferably not only useschanges in a characteristic, such as reflectivity, to track settling,but also includes sensing the absolute value of the characteristic,reflectivity, essentially immediately after a settling event occurs.

In yet another aspect of the present invention the improvement iscomprised of sensing and sampling a settling characteristic, such asreflectivity, in a stationary settling zone a spaced distance below theseparation boundary while settling of the separation boundary proceedsdown to said settling zone.

In still a further aspect of the present invention, the apparatus andmethod include a fluid plasma scanning assembly movably mountedproximate the settling tube and formed to scan a characteristic, such asreflectivity, of the plasma fluid in a scanning zone above theseparation boundary between the erythrocyte cells and the plasma fluid.

In the various aspects of the present invention, static or pulsedirradiation may be used. Radiation in the infrared frequency range ispreferred, but for some purposes, for example, white cell typing,radiation in the visible light range may be more advantageous. Usuallyreflectivity is the sensed and sampled characteristic, buttransmissivity of the test specimen also can be used.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graphic representation of a portion of an erythrocytesettling curve produced using prior art apparatus and methods.

FIG. 2 is a graphic representation of a tracking head displacement curvecorresponding to the settling curve of FIG. 1.

FIG. 3 is a front elevation view of an apparatus for determining thesettling characteristics of erythrocyte cells and white cellsconstructed in accordance with the present invention.

FIG. 4 is a side elevation view of the apparatus of FIG. 3 shownconnected to control and computation apparatus.

FIG. 5 is a top plan view of the apparatus shown in FIG. 3, with thecover removed.

FIG. 6 is a graphical representation of a tracking head displacementcurve using the apparatus of FIG. 3.

FIG. 7 is an enlarged, fragmentary, side elevation view, incross-section, of a schematic representation of the settling tube andsensing head of the apparatus of FIG. 3.

FIG. 8 is an enlarged, fragmentary, top plan view, in cross-section, ofa schematic representation of the tracking head and settling tube takensubstantially along the plane of line 8--8 in FIG. 3.

FIG. 9 is an enlarged, fragmentary, end elevation view of the sensinghead of the apparatus of FIG. 3.

FIG. 10 is a schematic representation of a control circuit used tocontrol motion of the sensing head in the apparatus of FIG. 3.

FIG. 11A is a computer display print-out of a settling curve generatedusing the apparatus of

FIG. 3 for a pregnant female in her first trimester.

FIG. 11B is a computer display print-out of an instantaneous erythrocytesettling rate curve based upon the curve of FIG. 11A.

FIG. 11C is a computer display print-out of a Fast Fourier Transform ofthe settling curve of FIG. 11A.

FIG. 11D is a computer display print-out of a Fast Fourier Transform ofthe settling curve of FIG. 11A with the first data point omitted.

FIG. 12A is a computer display print-out of a settling curve for anotherpregnant female in her first trimester.

FIG. 12B is a computer display print-out of an instantaneous settlingrate curve corresponding to the curve in FIG. 12A.

FIG. 12C is a computer display print-out of a Fast Fourier Transform ofthe settling curve of FIG. 12A.

FIG. 12D is a computer display print-out of a Fast Fourier Transform ofthe settling curve of FIG. 12A with the first data point omitted.

FIG. 13 is a computer display print-out of a settling curve for apregnant female in her second trimester.

FIG. 14 is a computer display print-out of an instantaneous settlingrate curve corresponding to the curve in FIG. 13.

FIG. 15A is a computer display print-out of a settling curve for apregnant female in her third trimester.

FIG. 15B is a computer display print-out of an instantaneous settlingrate curve corresponding to the curve in FIG. 15A.

FIG. 15C is a computer display print-out of a Fast Fourier Transform ofthe settling curve of FIG. 15A.

FIG. 15D is a computer display print-out of a Fast Fourier Transform ofthe settling curve of FIG. 15A with the first data point omitted.

FIG. 16A is a computer display print-out of a settling curve for afemale with a chronic fatigue condition.

FIG. 16B is a computer display print-out of an instantaneous settlingrate curve corresponding to the curve in FIG. 16A.

FIG. 17A is a computer display print-out of a settling curve for afemale who has lung cancer.

FIG. 17B is computer display print-out of an instantaneous settlingcurve corresponding to FIG. 17A.

FIG. 18A is a computer display print-out of a settling curve for afemale having breast cancer.

FIG. 18B is a computer display print-out of an instantaneous settlingrate curve corresponding to the curve in FIG. 18A.

FIG. 19A is a computer display print-out of a settling curve for a malesuspected of having Acquired Immune Deficiency Syndrome (AIDS) andhepatitis.

FIG. 19B is a computer display print-out of an instantaneous settlingrate curve corresponding to the curve in FIG. 19A.

FIG. 20A is a computer display print-out of a settling curve for a malehaving AIDS.

FIG. 20B is a computer display print-out of an instantaneous settlingrate curve corresponding to the curve in FIG. 20A.

FIG. 21 is a computer display print-out of a plasma fluid scan for thepatient of FIGS. 11A-D.

FIG. 22 is a computer display print-out of a plasma fluid scan for thepatient of FIGS. 12A-D.

FIG. 23 is a computer display print-out of a plasma fluid scan for thepatient of FIGS. 13 and 14.

FIG. 24 is a computer display print-out of a plasma fluid scan for thepatient of FIGS. 15A-D.

FIG. 25 is a computer display print-out of a plasma fluid scan for thepatient of FIGS. 16A-B.

FIG. 26 is a computer display print-out of a plasma fluid scan for thepatient of FIGS. 17A-B.

FIG. 27 is a computer display print-out of a plasma fluid scan for thepatient of FIGS. 18A-B.

FIG. 28 is a computer display print-out of a plasma fluid scan for thepatient of FIGS. 19A-B.

FIG. 29 is a computer display print-out of a plasma fluid scan for thepatient of FIGS. 20A-B.

FIG. 30 is a computer display print-out of a reflectivity measurementcurve for a pregnant patient in her first trimester.

FIG. 31 is a computer display print-out of a reflectivity measurementcurve for a female patient with breast cancer.

BEST MODE OF CARRYING OUT THE INVENTION

The blood sedimentation-based diagnosis apparatus and process of thepresent invention is based in part upon a principle found in the priorart, namely, tracking the separation boundary between erythrocyte cellsas they settle from the remaining plasma fluid. Prior separationboundary tracking apparatus and methods, however, have employedtechniques which inherently are incapable of generating data that can bereliably correlated to specific diseases and further introducedinaccuracies and noise into the tracking measurements so as to mask thedata obtained.

Prior Sedimentimeters--Detail

In FIG. 1, a section of a sedimentation curve 201 is shown which wouldbe generated by the apparatus and method of prior art U.S. Pat. No.4,041,502 to Williams et al. Actually, curve 201 has had its slopereversed from curve S in FIG. 3 of Williams et al., patent and it hasbeen drawn as straight line segments between a plurality of data points202-207.

The Williams et al. apparatus and method are based upon the sampling ofsensed data at fixed, predetermined time intervals, namely, every 15seconds. While the Williams et al. LED and photodiode operate or tensecontinuously, the sampling circuit looks at the total motor displacement(excursions), or samples sensed data, only at fixed time intervals of 15seconds.

As can be seen in FIG. 2 of the present application, Williams et al.apparatus has a light transmission threshold 209 above which thestepping motor is supposed to operate and below which the motor is shutdown. It is believed, however, that if the motor in Williams et al. isturned off at threshold 209, dropping of the boundary between red andwhite cells permits more light to pass through to the photodiode, whichturns the motor back on. There inherently is, therefore, some hysteresisin the servo circuit and stepping motor which effectively results in asecond or "motor on threshold" 210.

If a theoretical single threshold 209 truly existed, the stepping motorwould be continuously operating since the cells are settling virtuallyall of the time, but not necessarily at the same rate as the steppingmotor operates. The tracking head movement which is most likely to occurusing the Williams et al. apparatus, therefore, is believed to be thesolid line stepped curve 211 in FIG. 2, which corresponds to the twothreshold light transmission curve 212. If one threshold 209 truly werecontrolling, broken line curve 213 would illustrate the motion of theWilliams et al. tracking head. In either case, the straight line segment214 between data points 202 and 203, which segment forms curve 201 inFIG. 1, does not reflect the actual displacement of the tracking head.

Each of segments 214-218, in Williams et al., therefore, is anapproximation of the tracking head motion, which approximation does notreflect the sedimentation which is occurring between the data pointstaken every 15 seconds. Moreover, and very importantly, the selection orsampling of data in Williams et al. is driven arbitrarily. Data points202-207 only coincidentally (and rarely) are taken at times whensignificant settling rate changes are occurring.

As may be seen from FIG. 2 of the present application, the Williams etal. curve segment 214 completely misses the knee and slope reversalpresent in more accurate curve 213, and for that matter in stepped curve211.

During a one hour test, the Williams et al. apparatus generates a totalof only 240 data points. Obviously, the approximation which is thesettling curve generated by the Williams et al. apparatus becomes evenless accurate when the first derivative is calculated. The arbitrarilyselected data points are used to compute a change in height, Δh, whichis then divided by the change in time, Δt, during which the heightchange occurred. In Williams et al., Δt is always 15 seconds.

In Williams et al., therefore, the instantaneous settling rate or firstderivative curve is, in fact, only a rough approximation or amacro-curve which generally resembles the shape of an accurate slopecurve, but clearly is not capable of accurately illustrating themicro-curves (knees and slope reversals) which are actually occurringbetween data points.

Separation Boundary Tracking

In a first aspect of the present invention, therefore, a much moreaccurate settling curve and instantaneous settling rate curve areobtained by sensing at a very high rate and sampling sensed data whensignificant settling occurs.

As used in this application, the expressions "sensing" and "sensed"shall mean the receipt of a signal from the test specimen indicating acharacteristic of the specimen, for example, a signal indicating theamount of reflectivity of the specimen to infrared radiation. Thus, thereflectivity level of the specimen may be sensed every 10 milliseconds,but not necessarily sampled or measured. The expressions "sampling" and"sampled" shall mean one or both of: (i) the sensed characteristic isstored, recorded or captured, or (ii) the sensed characteristic is usedto store, record or capture related data to the sensed characteristic,such as, the time at which the sensed characteristic exceeded or fellbelow a threshold level. The expression "measured" shall mean that theabsolute value of the sensed characteristic has been stored or recorded.

Thus, the amount or sensed quantity of the reflectivity is recorded, asopposed to sampling or recording of related data, such as the time. Inthe sedimentation sensing apparatus and process of the present inventiondata sampling or capture is driven by significant settling of theerythrocyte cells, regardless of the timing of such settling, while inthe prior art data sampling or capture was driven by time, regardless ofthe settling activity. Settling-driven data capture is achieved in thepresent invention by sensing data at a rate which is much higher thanthe occurrence of significant red cell settling, and sampling inresponse to the sensed characteristic when significant settling occurs.This approach allows the present apparatus and method to capture themicro-curve data which has not previously been available for use incorrelating curve patterns with inflammatory conditions.

Other, probably second order, problems are present in prior art systemssuch as Williams et al., which employ the transmission of light throughthe test specimen in order to effect tracking. Such instruments areoperating constantly above the separation boundary or meniscus betweenthe settling erythrocyte cells and the plasma fluid, because the motoron threshold requires transmission of light through the specimen abovethe separation boundary. Data taken in this area, however, is subject tosignificant masking phenomena.

The formation and breakdown of rouleaux, with associate relative upwardmigration of plasma and white cells, can produce micro-turbulence at theseparation boundary in which red cells are carried temporarily up intothe plasma. These red cells reduce the transmissivity of the specimenabove the separation boundary. Additionally, the separation boundarytends to have an upwardly concave meniscus. Thus, transmissivity can beinfluenced by the surface tension or the meniscus at the separationboundary.

Still further, the white cells immediately above the separation boundaryalso are settling, and light or radiation transmission through theplasma fluid containing white cells at and above the separation boundarycan vary, not as a function of erythrocyte cell settling, but as afunction of settling of the white cells contained in the plasma fluid.

The combined masking effect probably is not significant in the Williamset al. apparatus and method because the long sampling interval has aneven greater masking effect. However, when more accurate tracking isundertaken, the use of a method based upon transmission of radiationthrough the specimen above the separation boundary is believed to beinherently less accurate than sensing a characteristic below theseparation boundary.

In the sedimentation apparatus and process of the present inventiontracking of the separation boundary movement is achieved most preferablyby sensing the reflectivity of the erythrocyte cells, and in thepreferred form, the reflectivity to a static beam of infrared radiationis sensed. Thus, by employing an infrared beam, which is reflectedagainst the settling erythrocyte cells proximate and below theseparation boundary, an increase in reflectivity resulting from settlingof red cells just prior to and at the separation boundary is used tocontrol movement of the present tracking head. This process, therefore,can proceed without the tracking head ever "seeing" the plasma fluidcontaining white cells or reaching the upwardly concaved meniscus of theerythrocyte cells on the walls of the settling tube.

The sedimentation instrument and process of the present invention can bedescribed in further detail by reference to FIGS. 3, 4 and 5. A bloodcell settling measurement instrument, generally designated 21, isprovided for determining the settling of both red and white cells in atest specimen of blood. Mounted in measuring instrument 21 is a standardblood settling tube, 22, which is preferably a Wintrobe blood settlingtube (such as a model 6901 SEDITUBE having a 3.15 millimeter internaldiameter). It will be understood, however, that a Becton-DickinsonVACUTAINER settling tube, Westergren settling tube or other settlingtube also could be used in the present apparatus and process. Tube 22 isslidably received in collar 23 and a socket 24 in the instrumentframework or support stand. Securement means, such as a spring-biaseddetect assembly, or even a thumbscrew 26, secures the specimen tubefirmly in place in the instrument.

A blood specimen is prepared in a standard manner, corresponding to thetype of blood settling tube 22 employed in the instrument. Thepreparation of the test specimen will not be described herein and doesnot constitute a novel portion of the present invention. It iscontemplated, however, that the apparatus and process of the presentinvention can be used with blood specimens to which non-standardadditives have been added. For example, additives which accelerate redcell settling can be used for both red cell studies and white cellstudies.

Mounted proximate settling tube 22 is a sensing means, generallydesignated 31, which is formed to sense a characteristic of the testspecimen of blood during settling. In prior art apparatus, the sensingmeans sensed the transmissivity of the test specimen during settling.Sensing means 31 of the present invention preferably is formed to sensethe reflectivity of the specimen in an attempt to enhance further theaccuracy of the data gathered. As above set forth, it is believed thatradiation transmission through the specimen produces less accurate data.It will be understood, however, that settling-driven data capture orsampling also would yield significantly enhanced settling andinstantaneous settling rate data, as compared to prior art instruments,even if the present instrument employed transmissivity through thespecimen instead of reflectivity off the specimen. In the broadestaspect of the invention, therefore, the characteristic sensed can betransmissivity, reflectivity, or another characteristic which changesduring settling of the erythrocyte cells, as long as data capture isdriven by settling and the sensing rate is sufficiently high relative tothe settling rate to insure sampling when significant settling occurs.

Sensing means 31 is most preferably mounted to a movable sensing ortracking head 42 and movement of tracking head 42 is, in turn,controlled by control means that is responsive to signals from thesensing means. The control means causes the tracking head to follow theseparation boundary between erythrocyte cells and plasma fluid duringsettling of the red cells in the settling tube. Control means of thepresent invention preferably includes a stepping motor 32 which iscoupled electrically by conductor means 33 to a control circuit 34coupled to computer processing unit (CPU) 36 (FIG. 4). Signals fromsensing means 31 are transmitted by conductor means 37 to controlcircuit 34 and from the control circuit to CPU 36 in a manner describedmore fully hereinafter.

Stepping motor 32 drives a drive pinion 39 which is matingly engagedwith a rack 41 that is movably mounted to linear bearing 40. Raising andlowering of tracking head 42 is the result of displacement of rack 41 onwhich the sensing means 31 is mounted (for example, by retainer member43). The storing or recording of the settling data and the approximateinstantaneous settling rate data is accomplished by CPU 36, which actsas sampling means responsive to at least one of the sensing means andcontrol means to sample and store data and calculate the approximateinstantaneous settling rate.

It should be noted that in the present invention the approximateinstantaneous settling rate which is calculated is not strictly thefirst derivative of the settling curve. Instead, the present apparatusand method calculate a "divided difference," which is equal to a changein height of the separation boundary divided by the time difference orinterval in which the change in height occurs. As will be set forth inmore detail below, the change in height is a constant, k, in thepreferred embodiment of the present invention, and the time difference,or the time interval during which the constant height change occurs, isa variable. Thus, the approximate instantaneous settling rate is adivided difference curve, namely, Δh/(t₂ -t₁) or k/Δt. In the improvedcell settling instrument of the present invention, sensing means 31 andcontrol circuit 34 are formed to sense a settling characteristic, suchas reflectivity, at a very high rate and to sample and/or record thatcharacteristic or the time data when significant settling occurs.

Computer 36, through control circuit 34, senses the level orreflectivity output of sensing means 31. As erythrocyte cells settle,the reflectivity of the specimen proximate and below the separationboundary between plasma containing white cells and the red cells startsto increase, i.e., there are fewer red cells to absorb the infrared beamand proportionately more white cells and plasma, and thus, more of thebeam is reflected. As the reflectivity of the specimen increases to apredetermined threshold, for example, between about 5 percent and about20 percent above a calibrated reflectivity level of the specimen, theCPU, through control circuit 34, actuates stepping motor 32 and trackinghead 42 is lowered which, in turn, lowers the sensed reflectivity. Atthe same time, the CPU stores or samples the time at which thereflectivity exceeded the predetermined threshold and the position dataof the tracking head similarly is correlated to the time data which hasbeen sampled.

The operation of stepping motor 32 and control circuit 34 to achievesettling-driven data capture can best be understood by reference to FIG.6. In FIG. 6, data points 202 and 203, as well as connecting curvesegment 214 from FIG. 2 for the prior art Williams et al. apparatus areshown. In the reflectivity-based system of the present inventionstepping motor 32 is actuated for one step any time the reflectivityrises to a "Motor On Threshold" level 220. Data point 202 occurs when astep of the stepping motor is completed and the reflectivity sensed bysensing means 31 is at point 221 below threshold 220.

As the erythrocyte cells settle, the decreasing density of theerythrocyte cell proximate the approaching separation boundary 48results in an increasing sensed reflectivity until point 222 is reachedat the threshold level 220. When level 220 is reached, CPU 36 stepsmotor 32 by one step, which reduces the sensed reflectivity again to alevel 230 below threshold 220. This process is repeated over and overduring the full settling period.

At each step taken by the stepping motor, the time at which the stepoccurred is stored or captured in CPU 36 and provides a data point forthe settling and divided difference curves of the present invention.Thus, in FIG. 6, there are 10 data points between data points 202 and203, and the micro-curve 213 can be drawn between these points to muchmore accurately track the erythrocyte settling than the simplistic curvesegment 214.

In the present invention, therefore, the data sampling means 36 isresponsive to a change in a sensed settling characteristic, not anarbitrary time interval, to capture, sample or record data. The settlingcharacteristic sensed by sensing means 31 is reflectivity of thespecimen and this characteristic changes with specimen settling.

If the rate of sensing the settling characteristic is high relative tothe occurrence of a measurable or significant amount of settling, thedata sampled will be relatively accurate. It is not currently knownprecisely how much faster the sensing rate should be than the measurablesettling rate to produce data that is sufficiently accurate to enablethe correlation of settling to inflammatory conditions, but sensing at arate of about 50 times the maximum expected settling rate forerythrocyte cells appears to produce data which is sufficiently accuratefor correlation. It may be, however, that a sensing rate of ten times oronly three times faster would suffice, particularly as the size of thesteps is decreased.

In the apparatus of the present invention reflectivity sensing rateshave been employed in the range of once every 0.01 to 0.002 seconds. InFIG. 6 vertical lines 223 represent such sensing times, and it will beseen that reflectivity is increasing from point 230 toward level 222 ateach sensing time 223. Stepping motor 32 in the illustrated apparatussteps sensing means 31 down by 0.034036 millimeters for each step. Usingthe present invention, maximum settling rates of erythrocyte cells highenough to produce stepping of motor 32 twice in one second have beenobserved. The maximum rate of erythrocyte cell settling, therefore, maybe about one 0.034036 millimeter step every 0.5 seconds, or about 0.068millimeters per second.

A sensing rate of once every 0.01 seconds, therefore, is 50 times fasterthan a settling step would occur under maximum settling conditions, anda sensing rate of once per 0.002 seconds is 250 times faster thansettling sufficient to cause the motor to take a step.

The sensing rate, therefore, should be sufficiently high, relative tothe maximum expected settling rate, that the sensed settlingcharacteristic will be sampled at essentially the same relative timeduring settling. Thus, time data will be sampled or stored each time thereflectivity reaches level 220 plus or minus 0.01 seconds, which willgive a high degree of confidence that separation boundary is in the sameposition relative to sensor 31 for each time a step is taken.

Using the example of FIG. 6, the method and apparatus of the presentinvention would generate 12 data points in 15 seconds and a 0.41millimeter drop of separation boundary 48, while the apparatus of theWilliams et al. patent would generate two data points. Moreover, thedata points using the present apparatus increase in number duringperiods of rapid settling and are captured when settling occurs, not atpredetermined time intervals.

The divided difference curve generated using the present apparatus isbased upon Δh/(t₂ -t₁). While Δh=k=0.034036 millimeters, Δt₁ is notnormally equal to Δt₂ and may vary substantially. The divideddifference, or approximate instantaneous settling rate, data based uponmicro-curve 214, therefore, also is much more accurate. In FIG. 6, forexample, the slope of curve 214 is 0.41/15=0.0273 mm/sec. By contrast,the slope of curve 213 at segment 228 is 0.034036/.75=0.0454 mm/sec.

In addition in the improved sedimentation tracking instrument of thepresent invention, sampling means 36 is responsive to sensing means 31to maintain the sensing means located in a movable tracking zone whichis centered proximate and below separation boundary 48 up to a positionat, but not above, the separation boundary. The sensing means in thepresent invention is sensing a change in the characteristic of the testspecimen in a tracking zone which includes the settling erythrocytecells, rather than operating in the plasma above the erythrocyte cells.

Tracking Head

As best may be seen in FIG. 7, blood specimen 46 is placed in settlingtube 22. Initially the specimen is a mixture of erythrocyte cells, whitecells and fluid plasma which is substantially uniform and unsettled andextends to level 47 in the settling tube. As settling begins, aseparation boundary 48 between the settling erythrocyte cells 49 and therelatively, but not completely, clear plasma fluid 51 begins to occur.As the separation boundary sinks or drops down tube 22, the controlcircuit and CPU in the instrument of the present invention positionsensing means 31 in a tracking zone, Z, which extends from a position 52below separation boundary 48 to a position 53.

In FIG. 8, a beam or band of static radiation 56 can be seen to betransmitted from transmitter means 57 through tube wall 22 until itmeets specimen 46 in tracking zone Z. The beam then is reflected fromspecimen 46 as reflected beam 58. A portion of reflected beam 58 passesout of tube 22 to reflectivity detector means 59. Part of beam 56 isreflected at surface 50 of specimen 46 and part is absorbed and/orreflected out of the specimen inwardly of surface 50. Control circuit 34and CPU 36 sense the amount or the intensity of the reflected beam anddetermines whether or not to lower rack 41 and tracking head 42. In thepreferred form, angle α of transmitter 57 from a plane 60 through thecenter of the tube is equal to about 20°, although it may convenientlyfall in the range of about 10° to about 40°, and angle β ofreceiver/sensor 59 is about equal to the angle α on the other side ofplane 60.

As illustrated in the drawing, radiation 56 is a static beam of infraredradiation. It is believed that radiation pulses can be used to increasethe sensitivity of the present instrument even further. In a pulsedradiation system narrow band peak-to-peak measurements of thereflectivity are sensed by a synchronous detector. This approach alsomay make simultaneous tracking and scanning possible using multiplesensing heads and pulses of different frequencies so that spurioussignals or noise from the other transmitter can be filtered out.

The operation of control circuit 34 and CPU 36, therefore, is toposition the reflectivity transmitter/sensor assembly 31 at the trackingzone (FIG. 7). As sedimentation occurs, sensor 31 monitors thereflectivity of the mixture of plasma fluid and erythrocyte cells in thetracking zone. During this process separation boundary 48 drops to thedotted line position in FIG. 7. At or about the time separation boundary48 reaches beam level 52, the reflectivity of the test specimen willincrease by reason of a significantly decreased density of erythrocytecells. This reflectivity increase occurs even though there areerythrocyte cells above the sensor. The control circuit and CPU,detecting an increase in reflectivity from sensor 31 above predeterminedthreshold 220 (FIG. 6) actuates motor 32 to step tracking head 42 downby one step to level 52a, which moves the tracking zone Z down to a newtracking zone, Za. While exaggerated in FIG. 7 for the purpose ofillustration, tracking zone Z in the preferred form has a height equalto the height of the emitter slit opening 104 as defined by masking 120(FIG. 9). In the embodiment of the drawing opening 104 has a heightdimension of about 1 millimeter. Thus, one step by motor 32 movestracking zone Z down by 0.034036 millimeters, which is only about 1/30of the height of the tracking zone Z. This stepping process is repeatedover the entire settling period, and enables the apparatus to generate asettling curve and enables CPU 36 to calculate the approximateinstantaneous settling rate or the divided difference curve.

Control Circuit

One form of control circuit 34 which is suitable for use with theapparatus and process of the present invention is shown in FIG. 10.Infrared red cell sedimentation sensor 31 and a white cell scanningsensor 81 (the use of which is described below) can be seen to becoupled through amplifiers 91 to analog-to-digital converters 92a and92b. Converters 92a and 92b transform the analog reflectivity signalreceived by infrared photodetectors 93 and 94 into 8-bit signals thatare transmitted to buffer 96 and subsequently to CPU 36.

The selection as between sensor 31 and sensor 81 is controlled bysignals from CPU 36 through address decoder 98, which in turn is coupledthrough flip-flop 97 to the analog-to-digital converters so as to permitpassage of one of the signals to buffer 96. As will be understood, acircuit also could be provided for simultaneous transmission andrecording of data from both detectors 93 and 94. As above noted,however, simultaneous transmission by two transmitters probably willrequire pulsed signals at sufficiently differing frequencies thatdetectors 93 and 94 do not mistakenly detect spurious signals from thewrong emitter.

Similarly, CPU 36 communicates with address decoder 98 to driveflip-flop 99 so that a selected one of infrared emitter 101, for redcell settling sensing, and the white cell scanning infrared emitter 102is actuated. The flip-flop 97 activates the analog-to digital converter92 which corresponds to the actuated one of emitters 101 and 102.

When the reflectivity increases by a predetermined amount (e.g., 18%)above a calibrated level stored in the CPU for each tracking position,as set forth below, the CPU further signals the address decoder 98 toactuate motor controller 103 for operation of one of motor drives 104and 106, which steps one of the stepper motors 32 and 86 by a singlestep.

The infrared emitters and detectors used in implementing the process andapparatus of the present invention can be an infrared LED and a phototransistor, as for example, are manufactured by Vactec, Model No.VTR17D1, or a pin photodiode can be substituted for the phototransistor. As can be seen in FIGS. 8 and 9, the emitters and detectorspreferably are mounted in a housing having a mask 120 defining aslit-opening or window 104 which insures that a relatively narrow bandof radiation, e.g., 1 millimeter (0.04 inches) in height and 4millimeters (0.16 inches) in length, is directed against the testsample. Similarly, the detector preferably has a similarly formed narrowwindow 106, which reduces the likelihood of detecting spurious signals.The entire instrument is covered by a housing 105 which can be placedover and removed from instrument 21, and which has a black interiorsurface to reduce ambient signals sensed by detectors 93 and 94.

In order to provide sufficient sensitivity to small steps,analog-to-digital converters 92a and 92b, which normally operate with afive volt window, are voltage controlled to operate with a one voltwindow. Thus, receiver 93 would normally produce a digital count of 255from converters 92a and 92b as the voltage from receiver 93 varies fromzero to five volts. In circuit 34, converters 92a and 92b have beenscaled to have a zero count at about 2.5 volts and a 255 count at 3.5volts. Thus, each count of output from the converters 92 corresponds toa change in passed voltage of about 3.9 millivolts. Without voltagecontrol of converters 92a and 92b each change in output count wouldrequire about a 19.6 millivolt change in input. In instrument 21 a0.034036 millimeter change produced only a 3 or 4 count maximum changewhen a full five volt window is used, but changes by about 15 to about100 counts, depending on the color of the specimen, when a one voltwindow is used.

Calibration

The effect of using a very sensitive analog-to-digital converter windowis that changes induced by settling tube irregularities and differencesof color from blood specimen to specimen can introduce significant noiseinto the data. In order to set the stepping motor threshold 220, andthereby produce uniform and repetitive actuation or stepping of thetracking head for various specimens, it is an important feature of thepresent invention that the reflectivity for each test specimen of bloodand the settling tube be calibrated. The reflectivity of blood variesconsiderably from patient to patient, making it essential that a meansfor calibration of instrument 21 be provided.

Calibration of the reflectivity of red analog-to digital converter 92afor color is accomplished through the following process. Control circuit34 steps the reflectivity sensor assembly 31 up from a positionproximate collar 23 one step at a time while sensing the reflectivity ofthe combined tube and specimen.

As will be seen in FIG. 10, receiver 93 is connected to differentialamplifier 91a, which in turn has digital-to-analog converter 126aconnected thereto and driven by CPU 36. At each step up tube 22 fromcollar 23, CPU 36 varies the offset calibration voltage of converter126a so as to produce a constant or set output voltage, V_(set), in FIG.6. In the preferred form V_(set) is equal to 2.696 volts, which isequivalent to a count of 50 on converter 92a. Thus, at the various stepsover the scanned height of tube 22, CPU 36 adjusts the offsetcalibration voltage to produce a constant output voltage to theconverter 92a and the digital input required at each position along thetube to produce the constant voltage is stored in memory in the CPU.SINCE CPU 36 is coupled to the stepping motor assembly, the CPU alwaysknows the position of the sensing heads and can use that positioninformation to select from memory the correct input to converter 126a.This calibration removes any differences in reflectivity along the tubedue to tube irregularities and also removes variations in reflectivityand changes of color from specimen to specimen. Voltage threshold 220(FIG. 6) is set at a level which is sufficiently above V_(set) to insurethat the sensed changes in reflectivity are due to settling and notother phenomenon, such a slight color change during aging. Empiricallyelevating threshold 220 to be between about 5 to about 20 percent aboveV_(set) seems to provide a threshold above the calibrated reflectivityvoltage which causes reflectivity changes to indicate settling.

The voltage threshold 220, for example, may be set to be 150 countsabove the V_(set), namely, at 3.284 volts, which is 18 percent aboveV_(set). Thus, the converter 92a generally will be operating at a countlevel of 200 and below.

Starting the Test

Once the reflectivity calibration for the full height of the tube isstored in CPU 36, the threshold level 220 can be used to locate the topof the specimen and to start the test. Tracking head 42 can be raiseduntil the sensed reflectivity exceeds level 220, which indicates thattop 47 of the specimen has been reached.

Circuit 34 then drops the tracking head down by a small amount, forexample, 2.5 mm, and begins to sense and store reflectivity of the testspecimen at a fixed location. Sensor assembly 31 is maintained at 2.5 mmbelow the beginning separation boundary height, and the settling testbegins when reflectivity increases over threshold 220, or when 3 minuteshave elapsed, whichever is earlier. The tracking head then tracks orfollows the separation boundary down tube 22, and the time of eachchange in position of sensor 31 is stored in a data file in CPU 36.

While radiation in the visible light frequency range is believed to besuitable for reflectivity tracking of boundary 48, the preferred form ofthe apparatus and process employs infrared radiation. It is believedadditionally that the process of the present invention also could beperformed using photo-optic or fiber optic or laser-driven arrays ofsensors to sense at a sufficiently high rate to give enough discretevalues of reflectivity in each zone of the settling tube for thesettling period (e. g., one hour) to enable correlation with specificinflammatory conditions. It is believed that use of a fiber optic arraymay enable approximation of a full-height real-time analysis of theentire settling tube.

Utilization of Data

Since the instrument and process of the present invention producesettling curves and divided difference curves which are much moreaccurate than previously was possible, it is believed that patternanalysis techniques will enable correlation of patterns with specificinflammatory conditions. More particularly, it is believed that acomputer implemented neural network will be able to compare sensedspecimens with stored "learned" data to match specimen patterncharacteristics with known pattern envelopes for inflammatoryconditions. Limited clinical testing to date indicates a stronglikelihood that distinctive patterns exist.

As will be seen from the Examples below, patterns in divided differencedata, or the approximate instantaneous settling rate, seem to reoccur.Additionally, Fast Fourier Transforms of the settling curve data alsoyield frequency distribution patterns of the frequencies which make upthe settling curve that appear to be reoccurring.

The apparatus of the present invention, in fact, resulted in newclinical testing of a patient who originally had not been diagnosed ashaving Acquired Immune Deficiency Syndrome (AIDS). The patient's divideddifference curves strongly suggested the possibility of AIDS, and newclinical evaluation confirmed the presence of the disease.

As also may be seen on the Fourier Transform displays, the word "LOWS"appears, followed by a number. The number indicates the number of timesthe frequency distribution curve reaches a low point or minimum.Preliminary statistical analysis indicates that the LOWS number can beused with the number of data points taken to produce a Pathology Indexwhich is a better indicator than the ESR as to the degree of sickness ofthe patient. If the LOWS number is divided by the number of data points,and the result is multiplied by 100 and subtracted from 100, an indexnumber results in the range of 0 to 100 in which patients at the highend of the index are very sick.

Time-Driven Sampling Approximation

Once accurate settling curves have been obtained, it becomes apparentthat a measurable drop, i.e., one step, in separation boundary 48 occursabout once every one-half second during rapid settling periods.Accordingly, an approximation of the settling curve can be obtained bytime-driven sampling.

If small steps are taken and data is sampled at least twice per second,the resulting settling curve will begin to approximate a sensing drivensampling curve. Thus, for 0.1 millimeter steps, or less, and 0.5 secondsampling rates, or higher, the resultant data begins to approximate themore accurate sensing driven data. If the steps are 0.068 millimeters(twice the preferred step size) and the sampling is taken every 0.05seconds (10 times the fastest expected settling for one step), a goodapproximation may result, but it will have two significantdisadvantages. First, it is still an approximation. Second, it requiresthe storing of much more data.

Scanning Erythrocyte at Fixed Locations

In addition to tracking separation boundary 48, it has been discoveredthat by sensing reflectivity at a fixed location a significant distancebelow the separation boundary a substantial decrease and then increasein the reflectivity can be measured. This dip in the absolute ormeasured value of reflectivity is observed at the start of separationboundary tracking.

As above described, sensing head 31 is positioned at 2.5 millimetersbelow top 47 of the specimen at the start of tracking. If the absolutevalue of reflectivity is sampled at that fixed location, for example, atabout 0.5 second intervals, or faster, a decrease in reflectivity,followed by an increase in reflectivity, is measured.

It is hypothesized that this phenomenon also may be useful incorrelation with inflammatory conditions. Thus, there may be a movingband of denser erythrocyte cells and/or rouleaux which precedes boundary48 and settles down tube 22. By sensing and sampling at a fixed locationa significant distance below the separation boundary the passage andcharacteristics of that moving, more absorbent, band of cells can bedetermined.

By way of an example, in a two sensing-head system, such as is providedby sensing head 31 and sensing head 81, tracking sensor 31 can be usedto sample or measure reflectivity for a first 2.5 millimeter step belowtop 47, and second head 81, operating at a different frequency and usingpulsed infrared radiation to avoid feedback to sensor 31, can be steppedto a position 5.0 millimeters below top 47. Once the separation boundaryreaches first head 31, it will track the separation boundary as abovedescribed. When boundary 48 reaches second head 81, it will berepositioned by CPU 36 by stepping it down by a significant distance,such as 2.5 millimeters. At the new fixed location head 81 will againsample sensed reflectivity changes to look for the movingradiation-absorbent band of erythrocyte cells.

This process will produce reflectivity measurements showing a series ofdips or depressed reflectivity patterns which should provide additionaluseful data as to blood settling.

At the present time the optimum increment below separation boundary 48which will reveal the reflectivity dip phenomenon is not known. It isobservable at steps of 2.5 millimeters, but a somewhat smaller incrementmay be acceptable. As used herein, however, the expression a"significant distance" shall mean a distance greater than the height ofthe sensor beam or pulse (which is currently about 1 millimeter).

It is also possible, although such phenomena have not been studied, thatother bands of reflectivity changes could exist at still lower levelsbelow boundary 48. Broadly, therefore, it is a feature of the presentinvention to scan the erythrocyte containing portion of the specimenfrom a fixed location a significant distance below boundary 48 at a highsensing rate and to sample reflectivity data, or time data, as settlingoccurs to attempt to measure or identify characteristic erythrocytesettling phenomena in the blood specimen below separation boundary 48.

It should be noted that as an alternative to measuring sensedreflectivity, such moving bands of cells also can be identified by timedata taken when the reflectivity starts to dip or decrease below athreshold, in a manner similar to tracking head 31 for tracking of theseparation boundary. Moreover, multiple thresholds can be used tocapture time and/or reflectivity data during passage of the band infront of sensor 81.

Reflectivity Measurement

As above described, changes in reflectivity resulting from erythrocytesettling are used to track the rate of descent of the separationboundary by capturing time data. It is an additional important featureof the present invention that a pattern of measured or absolute valuesof reflectivity be employed to correlate with inflammatory conditions.

In this process, reflectivity measurement is taken at the same relativetime to the settling of red cells, most preferably immediately after amotor step is taken. The high sensing rate above described causes thestepping head to be moved whenever the reflectivity exceeds threshold220. As can be seen from FIG. 6, however, the drop or decrease inreflectivity which occurs as a result of a single step can varysignificantly. Thus, the low points 230 can be seen to representdifferent levels of reflectivity which result when a single step of0.034036 millimeters is taken.

In the process and apparatus of the present invention, therefore, thefirst reflectivity measurement made after stepping the stepping motor(15 milliseconds) also is stored in CPU 36, and a pattern ofreflectivity measurements, as a function of the vertical position of thetracking head during settling, can be generated. Since the decrease inreflectivity resulting from a fixed downward step also providesinformation as to the rate at which erythrocyte cells are settling, thereflectivity measurement pattern also is believed to be susceptible tocorrelation to inflammatory conditions.

Plasma Scanning

Another aspect of the process and apparatus of the present invention isthat the white cells containing plasma above the settled erythrocytecells can be scanned to produce data which is also useful in diagnosisof inflammatory conditions. More particularly, the apparatus and processof the present invention include means for scanning plasma fluid 51 tosense a characteristic of the plasma fluid which provides data that canbe used alone or together with other erythrocyte settling data.

As set forth above, settling instrument 21 preferably includes a secondsensing means 81 mounted on a second tracking frame 82. Mounted tolinear bearing 85 for vertical reciprocation is rack 83, and a pinion 84is matingly engaged with the rack and driven by a second stepping motor86 (FIG. 5). Sensor 81 also preferably is an infrared sensing assemblyhaving a transmitter and an infrared detector/receiver constructed asdescribed in connection with sensing means 31. Sensor assembly 81 can besecured to scanning head 82 by retainer washer 87. Both sensor 81 andstepping motor 86 are coupled to control circuit 34 and CPU 36 byelectrical connector cable means for communication of control signalsand sensing signals therebetween.

Instead of stepping down with the separation boundary 48, however,sensor 81 is not employed until after a significant amount of settlingof erythrocyte cells has occurred. Most preferably, sensor 81 isemployed after the one hour settling period, during which sedimentationrates are recorded using sensor 31. It is possible for sensor 31 to beused to perform both the sedimentation tracking and the plasma fluidscanning, if these functions are done sequentially, but having twosensor heads allows overlap in the sensing functions, if needed ordesired, as long as feedback is prevented by frequency discrimination ora similar technique. It also is possible to drive sensors 31 and 81 by asingle motor if plasma scanning is done after separation boundarytracking. The use of two sensors has the advantage of allowingcalibration of red cell sensor 31 to be very precise, while thecalibration of white cell sensor 81 can be legs precise. It is alsopossible, however, to use a single sensor 31 and have CPU 36 recalibratesensor 31 for plasma scanning after erythrocyte tracking is completed.

In the preferred process, once the erythrocyte sedimentation trackingand erythrocyte reflectivity measuring has been completed, controlcircuit 34 will cause scanning of plasma 51. Plasma scanning will beundertaken only if the one hour erythrocyte settling rate (height drop)is over a predetermined minimum level. Circuit 34 brings scanning head82 up to the top 47 of specimen 46. Control circuit 34 will thereafterstep tracking head 82 and sensor 81 down toward separation boundary 48of the specimen. At each step, again preferably 0.034036 millimeters,the reflectivity of the plasma fluid will be sensed and sampled orrecorded. This is continued until boundary 48 of the specimen isreached, and a record of the position and plasma reflectivity during theentire scan is recorded in the CPU memory. The scan data, for example,can be displayed on output device 62.

Stepping from top 47 to boundary 48 preferably is accomplished in oneminute, and then the process is repeated. Head 82 raises to top 47 andreflectivity is sensed at each step to boundary 48. CPU 36, however,averages the second set of sensed reflectivity data with the first, andthe scan again is accomplished in one minute. In the preferred processten one-minute scans are made and averaged into the data, and after thelast scan a plasma scan curve, such as those shown in FIGS. 21 through29 can be generated and displayed.

It is not possible, of course, to begin measurement of the reflectivityof plasma fluid 51 until the erythrocyte or red cells have settled fromthe plasma to a sufficient extent to provide a representative quantityof plasma. In practice this minimum threshold is an erythrocyte one hoursettling rate of about 7 millimeters.

Scanning of plasma 51 is based upon the hypothesis that while theerythrocyte cells are settling, solids, such as various types of whitecells, in the plasma fluid also tend to settle. In some specimensbanding or layering of white cells can be seen in the plasma.Accordingly, scanning of the plasma also tends to produce reflectivitypatterns as a function of height or position below top 47. Study of thepositions of features such as the position of the maximum height, dipsin the reflectivity curve and the lowest value of reflectivity appear tohave indicated a high degree of correlation of such scan curve featureswith specific inflammatory conditions.

The white cell scanning sensor 81 also is preferably adjusted during theinitial red cell calibration scan of the specimen, but not to achieve afine calibration, but to set the output of analog-to digital converter92b to a count of 120. Since the plasma scan does not depend upon theprecise identification of the occurrence of settling, the converter 92bcan operate in the middle of the conventional five volt window, anddigital-to-analog converter 126b is used to set the output count at 120.Only one approximate setting is required, since currently the measuredvalues of the sensed reflectivity are not used in a quantitative manner.It will be understood, however, that the absolute values of white cellreflectivity may be determined to be useful. The current embodiment ofthe apparatus determines the position of reflectivity increases anddecreases relative to the separation boundary at the start of plasmascanning or top of the plasma. Once set for instrument 21, all scannedwhite cell curves will be scaled the same and can be compared to eachother and to stored data as to inflammatory conditions.

While plasma 51 can be scanned for reflectivity using infraredradiation, radiation in other frequency ranges and transmissivity mayalso be suitable for scanning and correlating a characteristic ofsettling of the plasma with inflammatory conditions.

More particularly, it is hypothesized that different types of whitecells tend to settle to differing degrees in the plasma. The bands inthe plasma which are sometimes visible to the naked eye tends to supportthis hypothesis. It is believed that colored light in visible frequencyranges may enable identification, and possibly even quantification, ofthe types and settling characteristics of at least some types of whitecells. Similarly, transmissivity measurements also may yield white celltyping and/or quantification data.

EXAMPLES

Using the apparatus and method of the present invention, blood specimensfrom various patients were studied and compared to clinical informationconcerning the patients.

ERYTHROCYTE SETTLING EXAMPLES Example 1--First Trimester pregnancy

FIGS. 11A-14D are computer print-outs of settling data for pregnantpatients during various stages of pregnancy. Since pregnancy includesinflammatory conditions as an aspect of a normal pregnancy, erythrocytesettling and white cell scanning can be used to monitor pregnancy forabnormalities.

In FIGS. 11A-11D a female patient 24 years of age and in her firsttrimester of the pregnancy has been tested using the apparatus andmethod of the present invention. This same patient's white cell scan canbe seen in FIG. 21.

FIG. 11A shows a one-hour Erythrocyte Settling Rate, "ESR" of 14.3millimeters, which is a relatively low settling rate suggesting minimalinflammatory process. Sedimentation curve 121, however, appears to havea constant slope and is not very instructive or helpful in assessing thepatient condition. In FIG. 11B, a divided difference or approximatesettling rate curve 122 is shown. As will be seen, this curve now startsto reveal patterns in the data, not readily apparent from curve 121. InFIG. 11C a Fast Fourier Transform 123 of the settling data in curve 121has been performed in an attempt to obtain a mathematical analysis ofthe distribution of frequencies making up settling curve 121. As will beseen, Fourier transform curve 123 shows patterns which are verypronounced and appear to repeat in many areas. Finally, curve 124 inFIG. 11D is a Modified Fast Fourier Transform of the data of curve 121.The analysis differs from FIG. 11C in that the first data point waseliminated. Again, however a very distinctive Fourier curve 124 results.

The use of Fourier transform curves is an attempt to facilitate patternrecognition from the accurate micro-curve data captured using thepresent apparatus by means of computer analysis. More particularly, itis believed that a Fourier transform of the settling curve data may lenditself to computer analysis by the use of neural network data processingtechniques. It is not known yet whether one or more of the divideddifference curve 122, the Fast Fourier Transform curve 123, or theModified Fast Fourier Transform curve 124 will be the most productive touse with a neural network to achieve correlations of curves withspecific inflammatory conditions.

Even without the precision of computer analysis of the patterns producedby the instrument of the present invention, one can visually identifyrepetitive characteristics in the various curves.

Example 2--First Trimester Pregnancy

FIGS. 12A-12D are four curves 131-134 corresponding to curves 121-124.Curves 131-134 are based upon settling data taken from a 23 year oldfemale also in her first trimester of pregnancy. As will be seen, thedivided difference curves 122 and 132 are very similar, as are the FastFourier Transforms. The peaks 126 and 136, for example, occur in almostexactly the same position, as is true of peaks 127 and 137 in curves 124and 134, and the shapes of these frequency distribution curves arealmost identical.

Since both of these pregnancies appear from clinical testing to beentirely normal, the possibility of using the settling data, whichclearly repeats, to develop a statistical model, or an envelope of datafor a normal first trimester pregnancy, seems quite high. Oncedeveloped, settling rate testing can be compared against the model andabnormal pregnancies identified.

Example 3--Second Trimester Pregnancy

In FIGS. 13 and 14 a 37 year old patient in her second trimester hasbeen tested. As will be seen the ESR has increased significantly fromthe first trimester pregnancies to 30.9 millimeters, but sedimentationcurve 141 is very similar in overall appearance to curves 131 and 121.

In FIG. 14, however, divided difference curve 142 is considerablyelevated and much more active than either of curves 132 or 122. FourierTransform curves similarly were different but are not shown only becausethe types of changes are also illustrated in the patient of FIGS.15A-15D.

Example 4--Third Trimester Pregnancy

A female patient 18 years of age and in her third trimester of pregnancywas the subject of the test of FIGS. 15A-15D. Again, sedimentation curve143 is not very instructive, but the divided difference curve 144clearly has recognizable differences as compared to curves 122, 132 and142. While the ESR for the third trimester patient, 35.9 millimeters,has not increased very much from that of the second trimester (5millimeters), the divided difference curve activity has increasedsubstantially.

It is recognized, of course, that there are factors, such as agedifferences, which may affect the curves, but the high coincidence ofsimilar curve features for curves 122 and 132 for patients ofsubstantially the same age tends to suggest that differentiation for agealso can be developed, if age affects the curves.

The Fourier curves 147 and 148 for the third trimester patient also arequite recognizably different from that of the first trimester patients.Interestingly, the maximum spikes are only slightly shifted, but theharmonic content has increased in FIGS. 15C and 15D, and patterns ofsecondary or harmonic frequencies are beginning to appear or be morerecognizable. Again, the possibility of developing an envelope of modeldata as a result of the improved accuracy of the instrument and methodof the present invention seems quite likely.

In FIGS. 16-20B patients with various inflammatory diseases were tested.

Example 5--Chronic Fatigue

FIGS. 16A and 16B are the settling rate curve 149 and divided differencecurve 150 for a female patient 37 years of age with a chronic fatiguecondition. Note that the ESR and settling curve are about the same asfor the 37 year old patient of FIG. 15A. The divided difference curves144 and 150, however, are recognizably different.

Example 6--Lung Cancer

The 59 year old female patient of FIGS. 17A and 17B has lung cancer.This patient's ESR is 57.6, which is symptomatic of substantialinflammation, and settling curve 168 and divided difference curve 169both are quite different from the previous curves. The activity ofdivided difference curve 169 in the early stages of settling is verysubstantial. It should be noted that curves 168 and 169 are based upon1692 data points, whereas a corresponding Williams et al. curve would bebased upon 240 data points. Moreover, in the area of rapid change at thestart of settling, the present instrument generates more data pointsthan at the less significant tail portion 170 of the curve. A computeranalysis of the micro-curves in the rapid falling first two-thirds ofthe curve would have over 1500 data points while Williams et al. datawould include 160 data points. An increase in data of almost 10 timescan be achieved with the instrument of the present invention, and thedata selection is settling-driven, not arbitrarily taken.

Example 7--Breast Cancer

FIGS. 18A and 18B show the settling curve 180 and divided differencecurve 190 for a 47 year old female patient with breast cancer. Thesettling rate is almost identical to the rate for the lung cancerpatient, and the settling curves 180 and 168 are quite similar. Thedifferences between the divided difference curves for these two diseasesagain are quite recognizable. In curve 190 there is less initialactivity and even more mid-range activity than curve 169 for the lungcancer patient. Curves 169 and 190 are clearly distinguishable from eachother and from the curves of the other Examples.

Examples 8 and 9--AIDS

The patients whose tests are shown in FIGS. 19A-B and 20A-B both haveAcquired Immune Deficiency Syndrome (AIDS). The first patient (FIGS. 19Aand 19B) is a 25 year old male without any observable complications. Thesecond patient is a 36 year old male who also has meningitis.

The ESR for curve 178 is almost identical to curve 149 (FIG. 16A) forthe patient with a chronic fatigue condition. The respective divideddifference curves 150 and 179, however, are clearly different, eventhough physicians believe that chronic fatigue virus and the AIDS virusare broadly related.

The effect of the complicating presence of meningitis appears toincrease the early activity (slope changes) in divided difference curvein FIG. 20B. The settling curve 176 also has a somewhat steeper slopewhich is reflected in the elevated divided difference curve. Again, thenumber of data points used to generate the first two-thirds of curves176 and 177 will be approximately 8 to 10 times the number employed inthe prior art.

PLASMA SCAN EXAMPLES

FIGS. 21-29 are examples of computer generated displays of plasma orwhite cell scans using the apparatus and process of the presentinvention. It was anticipated that there also would be variations in theplasma scan curves from individual to individual. Testing to datesuggests that the possibility of developing envelopes of plasma scancurves, which can be used alone or with settling data, and which have avery high degree of correlation to specific inflammatory conditions,seems high.

In FIGS. 21-24 are scans of the pregnant patients of Examples 1, 2, 3and 4. In FIGS. 21 and 22 both patients are in their first trimester,and they correspond to Examples 1 and 2, respectively. As will be seenby comparing these scans the overall shape of scans 151 and 152 is verysimilar. Both have peaks 153 and 154 followed by a dip which ends inshoulders 155 and 156. After the shoulders the curves drop dramaticallyat 157 and 158 to a minimum reflectivity (higher cell density proximateseparation boundary 48) at 159 and 160. Moreover, the relative spacingbetween these features has a high degree of consistency.

Both of these patients are having a normal pregnancy free ofinflammatory complications.

FIG. 23 shows the scan of the 37 year old patient in the secondtrimester of her pregnancy of Example 3. As compared to curves 151 and152, this curve 163 has a peak 164 which is much more closely followedby a very slight dip and shoulder 165. Curve 163 then descends rapidlyat 166 to an almost constant minimum or tail 167, which is longer byreason of the greater erythrocyte settling and more plasma to scan thanfor curves 151 and 152.

In FIG. 24, white cell scan 171 is for the 18 year old pregnant patientin her third trimester of Example 4. The peak 172, dip and shoulder 173structure has increased as compared to curve 163, but still can be seento be less than that of curves 151 and 152. Again, after shoulder 173there is a rapid drop 174 followed by a nearly level long tail 175.

Thus, white cell scans 151, 152, 163 and 171 show both a characteristicpregnancy curve, similarities and repeating structures for similarlengths of pregnancies and changes which differentiate the length ofpregnancy. All of these patients are experiencing normal pregnancies,but if they did have an inflammatory disease complication, the whitecell scans would be significantly altered.

In FIG. 25, for example, the white cell scan 181 for the 37 year oldfemale patient of Example 5 with a chronic fatigue condition is shown.The curve peaks at 182 and then dips slightly at 183. As will be seen,however, there is no shoulder after dip 183, nor is there a rapid dropto a minimum. Instead, dip 183 is the minimum and the curve continueswith a gradually rising long tail 184.

Comparing curve 181 with curve 171 makes it apparent that theinflammatory conditions of the two patients are radically different. Theone-hour erythrocyte settling rate for the chronic fatigue patient whosescan is shown by curve 181 was 37.4 millimeters, while the one-hoursettling rate for the third trimester pregnancy was 35.9 millimeters.Clearly, the erythrocyte settling rate alone is not a tool capable ofdistinguishing between these patients, but the white cell scan is.

In FIG. 26, scan 186 is for the 59 year old female patient with lungcancer of Example 6. Again, there is an initial peak 187, dip 188 andshoulder 189 structure. The spacing of these features is somewhatgreater than for pregnant patients, and while there is a drop 190, it isrelatively short. Curve 186 also includes a long tail 191 with a rise orshoulder 192 which is believed to be significant and is not present ineither the pregnant or chronic fatigue patients.

FIG. 27 shows a scan for the 47 year old female patient of Example 7 whohas breast cancer. Curve 195 includes a peak 196 and a shoulder 197,followed by a very short drop 198, an immediate rise 199 and a longdeclining tail 200. Thus, significantly different features can be foundbetween lung cancer scan 186 and breast cancer scan 195.

Finally, FIGS. 28 and 29 are plasma scan curves for patients with AIDS.Curve 301 is for the male patient who is 36 years old and curve 302 isfor the male patient who is 25 years old. As will be seen in bothcurves, there is an initial peak 303 and 304, but the peak is followedby greatly elongated dips or flat sections 305 and 306, which appears toterminate in very slight shoulders 307 and 308. After the shoulders 307and 308 the tails 309 and 310 tend to remain level or even risesomewhat.

Interesting similarities can be seen between AIDS scan 301 and chronicfatigue scan 181. This is not too surprising since both are believed tobe viral based with numerous similarities. When the divided differencecurves 150 and 190 are compared, however, these two patients can beclearly distinguished. Thus, the potential for use of a combination oferythrocyte data and white cell scans to enhance diagnoses also isapparent.

REFLECTIVITY MEASUREMENTS Example 10--Pregnancy First Trimester

FIG. 30 is a computer display curve 401 of reflectivity measurementsmade immediately (15 milliseconds) after each step during a settlingrate test for a pregnant patient in her first trimester. This patient isnot the patient of Examples 1 or 2.

In curve 401 the reflectivity measured at the zero abscissa point is atthe start of settling. This patient was 24 years old and her ESR was35.2 millimeters, which is high for a first trimester patient.

Example 11--Breast Cancer

In FIG. 31 a reflectivity measurement curve 402 for a female patient 39years old with breast cancer and an ESR of 51.4 millimeters is shown.This is not the same patient as Example 7.

Comparing curves 401 and 402 a clearly recognizable pattern differencein the measured reflectivity is discernable. The ability of the presentinstrument to take steps when settling occurs, to be settling driven,rather than time-driven, provides, it is believed, data in which thereflectivity measurements are more capable of being compared formeaningful differences.

Again, reflectivity measurements are believed to be useful alone, orwith settling data and/or plasma scans, to help in the correlation ofcurve patterns with inflammatory conditions.

What is claimed is:
 1. An apparatus useful in the diagnosis ofinflammatory conditions comprising:settling tube means formed to receivea test specimen of blood for settling of erythrocyte cells from plasmafluid; sensing means movably mounted proximate said settling tube meansand sensing changes in a characteristic of said plasma fluid; drivemeans coupled to said sensing means and driving said sensing means toscan said test specimen between a position proximate a top of said testspecimen and a position proximate and above a separation boundarybetween said erythrocyte cells and said plasma fluid; and means forrecording said characteristic coupled to said sensing means and formedfor recording said characteristic during scanning of said test specimen.2. The apparatus as defined in claim 1 wherein,said sensing means isformed to sense the reflectivity of said plasma fluid.
 3. The apparatusas defined in claim 1 wherein,said sensing means is formed to directradiation in one of the infrared and visible light frequency rangesagainst said plasma fluid.
 4. The apparatus as defined in claim 1wherein,said sensing means directs a beam of infrared radiation againstsaid plasma fluid and senses the reflectivity of said plasma fluid; andsaid means for recording, records reflectivity as a function of sensingmeans position.
 5. The apparatus as defined in claim 4 wherein,saiddrive means repetitively scans said sample; and said recording meansrepetitively records reflectivity as a function of sensing meansposition.
 6. The apparatus as defined in claim 5, andcomparator meanscoupled to said sensing means and responsive to said sensing means tocompare said reflectivity as a function of sensing means position forsaid test specimen with a reflectivity as a function of sensing meansposition for a control specimen of blood from a patient having a knowninflammatory condition.